The gram-negative bacterium Bordetella pertussis (B. pertussis), the causative agent of whooping cough, produces several virulence factors. Pertussis toxin (PT), the major virulence component of B. pertussis, appears to contain an important epitope that leads to the formation of antibodies capable of protecting against the disease. Therefore, PT has been extensively investigated with regard to its possible use in preparing vaccines for whooping cough (Black et al., 1988).
Pertussis toxin is a 105-kDa hexameric protein composed of five distinct non-covalently linked polypeprides designated (in the order of decreasing molecular weight) S1-S5.PT can be divided into two distinct functional units, the enzymatically active toxic A-protomer, consisting of a single polypeptide (S1), and the pentameric B-oligomer (S2, S3, two copies of S4, and S5, i.e. molar ratiol:1:2:1). The B-oligomer is responsible for binding of the toxin to the surface of eukaryotic target cells. The two S4 polypeptides form two distinct heterodimers with S2 and S3, which are in turn held together by S5 (see review by Gierschik, 1992).
The perrussis toxin gene has been cloned and sequenced (Nicosia et al., 1986; European Patent Application EP 0232 229; Locht and Keith, 1986; U.S. Pat. No. 4, 883,761). The individual subunits have been subcloned and expressed in E. coli in nonfusion form (Burnette et al.,1988) or as fusion proteins (Nicosia et al., 1987), and tested as antigens for protection against whooping cough.
The development of experimental autoimmune encephalomyelitis (EAE), as well as other autoimmune diseases in experimental animals, can be facilitated by injecting Bordetella pertussis concomitantly with inoculation of the autoantigen (Bernard et al., 1992). EAE is a neurological autoimmune disease which can be induced in experimental animals by a single injection of central nervous system (CNS) tissue homogenate or purified myelin antigens such as myelin basic protein (MBP) or proteolipid protein (PLP) in complete Freund's adjuvant (CFA) (Tabira and Kira, 1992). The clinical and pathological features of EAE are reminiscent of multiple sclerosis , and EAE is a well-accepted model for multiple sclerosis. In mice,. consistent elicitation of EAE was shows to be facilitated by administration of B. pertussis at the time of the encephalitogenic challenge (Munoz, 1985). Pertussis toxin (PT) was shown later to be the component of B. pertussis responsible for facilitating disease development, and it is now routinely used in place of B. pertussis for enhancement of autoimmune disease in experimental animals (Munoz, 1995).
In an analysis of the possible inununomodulating activity of various bacteria, it was found by the present inventor that B. pertussis not only enhances the development of EAE in mice, but can also protect against the disease depending on the time and route of injection (Lehman and Ben-Bun, 1993). The protective activity of B. pertussis was subsequently assigned to PT (Ben-Nun et al., 1993).
Pertussis toxin (PT), the major virulence determinant of B. pertussis , is composed of two distinct functional units: the A-protomer consisting of a single polypeptide (S1) that mediates adenosine diphosphate (ADP)-ribosylation of host G proteins, and the B-oligomer, a complex pentamer composed of subunits S2, S3, S4 and S5 in a respective molar ratio of 1:1:2:1, which mediates the binding of the toxin to host tissue by interaction with glycoproteins and glycolipids on many types of eukaryotic cells (Gierschik, 1992). PT was found to have mitogenic and immunoadjuvant properties (Munoz, 1985). The mechanism by which PT can enhance the development of EAE in mice is not yet clear. However, it has been suggested that PT facilitates the access of autoantigen-specific T cells to the CNS by affecting the vascular permeability of the blood-brain barrier (Linthicum et al., 1982).
The mechanism by which PT protects against the development of EAE is also unclear. To further understand how both enhancing and protective activities are mediated by the same, albeit complex, molecule, it is essential to delineate the regions of the PT holomer which may be associated with one or both of these activities.